{"files"=>["https://ndownloader.figshare.com/files/1016875"], "description"=>"<p>(<b>A</b>) THP-1 cells were pretreated with NF449 (10 µM), NF157 (50 µM), or KH7 (100 µM) for 30 min. (<b>B</b>) Treatment with NF157 was initiated 30 min before to 180 min after the treatment with LPS (10 µg/mL). At 24 h after LPS stimulation, supernatants were collected and the concentration of IL-6 was measured by ELISA. (<b>C</b>) Cells were pre-treated with NF157 for 30 min, and then incubated with 100 µM ATP for 24 h. Supernatants were collected and the concentration of IL-6 was measured by ELISA. (<b>D–E</b>) Cells were incubated with LPS (10 µg/mL) for 1–6 h (<b>D</b>). Cells were pre-incubated with NF157, and incubated with LPS for 6 h (<b>E</b>). At the end of incubation, mRNA level of IL-6 was determined by RT<sup>2</sup>-PCR as described under <i><a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059778#s2\" target=\"_blank\">Materials and Methods</a></i>. Each value represents the mean ± SE (n = 4–11). Significant differences between a test group and control group (LPS or ATP alone) are indicated by **(p<0.01) and ***(p<0.001), respectively.</p>", "links"=>[], "tags"=>["p2y11", "receptor", "antagonist", "lps-induced", "il-6", "thp-1"], "article_id"=>676448, "categories"=>["Medicine", "Immunology"], "users"=>["Hayato Sakaki", "Mitsutoshi Tsukimoto", "Hitoshi Harada", "Yoshinori Moriyama", "Shuji Kojima"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0059778.g004", "stats"=>{"downloads"=>2, "page_views"=>150, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effect_of_P2Y11_receptor_antagonist_on_LPS_induced_IL_6_production_in_THP_1_cells_/676448", "title"=>"Effect of P2Y11 receptor antagonist on LPS-induced IL-6 production in THP-1 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-04-05 01:47:28"}

{"files"=>["https://ndownloader.figshare.com/files/1016870"], "description"=>"<p>(<b>A</b>) THP-1 cells were transfected with 2 µg of two different shRNAs targeting SLC17A9 (No. 1 or No. 2) or scramble shRNA. Seventy-two hours after transfection, cellular membrane proteins were extracted and the expression of SLC17A9 was detected by immunoblotting as described in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059778#s2\" target=\"_blank\">Materials and Methods</a>. (<b>B</b>) THP-1 cells transfected with two different shRNAs targeting SLC17A9 or the negative control shRNA were stimulated with LPS. At 10 min after LPS stimulation, the supernatants were collected and ATP content was measured (n = 8–12). (<b>C</b>) The shRNA-transfected cells were incubated with LPS for 24 h, and then the concentration of IL-6 in culture medium was measured by ELISA (n = 10–14). Each value represents the mean ± SE. Significant differences between the control group (scramble) and the indicated group are indicated by ***(p<0.001).</p>", "links"=>[], "tags"=>["lps-induced", "atp", "il-6", "thp-1"], "article_id"=>676446, "categories"=>["Medicine", "Immunology"], "users"=>["Hayato Sakaki", "Mitsutoshi Tsukimoto", "Hitoshi Harada", "Yoshinori Moriyama", "Shuji Kojima"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0059778.g002", "stats"=>{"downloads"=>2, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_SLC17A9_is_involved_in_LPS_induced_ATP_release_and_IL_6_production_in_THP_1_cells_/676446", "title"=>"SLC17A9 is involved in LPS-induced ATP release and IL-6 production in THP-1 cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-04-05 01:47:26"}

{"files"=>["https://ndownloader.figshare.com/files/1016891", "https://ndownloader.figshare.com/files/1016892", "https://ndownloader.figshare.com/files/1016894"], "description"=>"<div><p>It is important to understand the mechanisms that regulate macrophage activation to establish novel therapies for inflammatory diseases, such as sepsis; a systemic inflammatory response syndrome generally caused by bacterial lipopolysaccharide (LPS). In this study, we investigated the involvement of extracellular ATP-mediated autocrine signaling in LPS-induced macrophage activation. We show here that ATP release via exocytosis, followed by activation of P2Y11 receptor, is a major pathway of the macrophage activation, leading to release of cytokines. Treatment of human monocyte THP-1 cells with LPS induced rapid ATP release from cells, and this release was blocked by knockdown of SLC17A9 (vesicular nucleotide transporter, VNUT), which is responsible for exocytosis of ATP. ATP-enriched vesicles were found in cytosol of THP-1 cells. These data suggest the involvement of vesicular exocytosis in the release of ATP. Knockdown of SLC17A9, the P2Y11 antagonist NF157 or knockdown of P2Y11 receptor significantly suppressed both M1-type polarization and IL-6 production in THP-1 cells, indicating an important role of activation of P2Y11 receptor by released ATP in macrophage activation. Next, the effect of NF157 on LPS-induced immune activation was examined <i>in vivo</i>. Administration of LPS to mice caused increase of serum IL-1ß, IL-6, IL-12 and TNF-alpha levels at 3–24 h after the administration. Pre-treatment of LPS-treated mice with NF157 suppressed both elevation of proinflammatory cytokines in serum and M1 polarization of peritoneal/spleen macrophages. Moreover, post-treatment with NF157 at 30 min after administration of LPS also suppressed the elevation of serum cytokines levels. We conclude that vesicular exocytosis of ATP and autocrine, positive feedback through P2Y11 receptors is required for the effective activation of macrophages. Consequently, P2Y11 receptor antagonists may be drug candidates for treatment of inflammatory diseases such as sepsis.</p> </div>", "links"=>[], "tags"=>["macrophage", "activation", "exocytosis", "atp", "p2y11"], "article_id"=>676457, "categories"=>["Medicine", "Immunology"], "users"=>["Hayato Sakaki", "Mitsutoshi Tsukimoto", "Hitoshi Harada", "Yoshinori Moriyama", "Shuji Kojima"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0059778.s001", "https://dx.doi.org/10.1371/journal.pone.0059778.s002", "https://dx.doi.org/10.1371/journal.pone.0059778.s003"], "stats"=>{"downloads"=>7, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Autocrine_Regulation_of_Macrophage_Activation_via_Exocytosis_of_ATP_and_Activation_of_P2Y11_Receptor_/676457", "title"=>"Autocrine Regulation of Macrophage Activation via Exocytosis of ATP and Activation of P2Y11 Receptor", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2013-04-05 01:47:37"}